Studying variants’ mutations overturns assumptions

Errors during the replication of the SARS-CoV-2 virus result in mutations in its genome. This produces variants.

Although mutations are constantly produced as SARS-CoV-2 replicates, only a small number of these mutations are responsible for an increase in transmissibility or disease severity.

Variants of concern, which have increased transmissibility, disease severity, or ability to evade the immune system, tend to carry multiple mutations in their genomes.

Experts have yet to understand the precise role of individual mutations in enhancing the transmission of the virus or causing more severe disease.

The Alpha variant, which was first identified in the United Kingdom in the fall of 2020, was 45–71{cfdf3f5372635aeb15fd3e2aecc7cb5d7150695e02bd72e0a44f1581164ad809} more transmissible than the wild-type SARS-CoV-2 that originated in Wuhan, China. Alpha was also associated with an increase in disease severity.

The Alpha variant had 23 mutations in its genome, including nine in the gene coding for the spike protein.

Scientists have observed multiple spike protein mutations in the different variants of concern, and some of these mutations are associated with increased transmissibility.

The spike protein is expressed on the surface of SARS-CoV-2, and it allows the virus to bind to the angiotensin-converting enzyme 2 receptor on the surfaces of human cells.

The cleavage of the spike protein at a specific site by the enzyme furin, which human cells express, is thought to be essential to facilitate the entry of the virus into airway epithelial and lung cells.

One mutation in the spike protein gene of the Alpha variant is at the furin cleavage site. Changes in the sequence of this site may influence the transmissibility of SARS-CoV-2.

The present study characterized the ability of this mutation at the Alpha variant’s furin cleavage site to influence the virus’ ability to infect and replicate in human cells.

To understand the impact of this particular mutation, the researchers first used a bioinformatics approach. Bioinformatics involves using software tools to analyze biological data, and it can predict the structure of proteins using genetic information.

The bioinformatics analysis predicted that the mutation at the furin cleavage site in the Alpha variant would slightly enhance the cleavage of the spike protein by the furin enzyme.

The researchers also conducted a biochemical assay in the laboratory to confirm this. They incubated a short fragment of the spike protein containing the furin cleavage site from the Alpha variant and wild-type SARS-CoV-2 with furin.

The assay showed that the spike protein fragment from the Alpha variant was cleaved to a slightly greater extent than the wild-type SARS-CoV-2, but only at a specific pH.

The researchers then used pseudoparticles to assess the impact of the mutation at the furin cleavage site on the ability of SARS-CoV-2 to enter human cells.

These pseudoparticles consist of a surrogate virus other than SARS-CoV-2 that expresses a specific SARS-CoV-2 protein. They have the essential components to infect a cell but cannot replicate, making them harmless.

In the present study, the researchers used pseudoparticles expressing the spike protein from wild-type SARS-CoV-2 and the Alpha variant.

The researchers also used a third type of pseudoparticle that expressed a modified form of wild-type SARS-CoV-2 spike protein that included one particular alteration found in the Alpha variant.

This modified wild-type SARS-CoV-2 spike protein only had one change at the furin cleavage site, which was caused by a specific mutation of the Alpha variant, but it did not have the other changes seen in the Alpha variant’s spike protein.

The researchers compared the ability of these three pseudoviruses expressing different spike proteins to infect Vero cells, which are laboratory-cultured kidney cells.

SARS-CoV-2 can enter cells by two main pathways. One involves the fusion of the virus envelope with the membrane of human cells and is mediated by an enzyme called TMPRSS2, which is on the surface of human cells. In the other pathway, the virus is engulfed by fluid-filled bodies called endosomes that are present inside the cells.

The researchers used two types of Vero cells. One expressed TMPRSS2 and favored entry by fusion. The other cell line favored the entry of the virus by the endosomal pathway.

There was no difference in the abilities of the pseudoviruses expressing the three different spike proteins to infect either type of Vero cells.

In other words, the presence of the mutation at the furin cleavage site of the Alpha variant’s spike protein did not enhance cell entry — through either the TMPRSS2 or endosomal pathways.

Since Vero cells are derived from the kidney, the researchers then infected laboratory-cultured cells from the human respiratory tract with the pseudoparticles expressing the three spike proteins.

The pseudoparticles expressing the spike from the Alpha variant were slightly more effective at infecting respiratory tract cells than those with the wild-type virus’ spike.

Interestingly, the pseudoparticles carrying the wild-type SARS-CoV-2 spike with the mutation at the furin cleavage site did not differ from the wild-type virus in their ability to infect respiratory tract cells.

The results of the experiment with respiratory tract cells suggest that mutations other than the one at the furin cleavage site are probably responsible for the increased capacities of the Alpha variant to transmit and to cause disease.

The researchers then compared the ability of samples of wild-type SARS-CoV-2 and the Alpha variant to replicate in the two Vero cell lines and human respiratory tract cells.

Similar viral titers were observed in all three cell types, suggesting that the Alpha variant did not replicate more quickly than wild-type SARS-CoV-2.

The SARS-CoV-2 spike protein is also known to mediate the fusion of infected cells with adjacent uninfected cells, thus facilitating the spread of the virus throughout the lungs.

Because there were no differences between the Alpha variant’s and wild-type SARS-CoV-2’s abilities to infect or replicate in human cells, the researchers decided to investigate any differences in the abilities of these spike proteins to induce cell fusion. The researchers found that the Alpha variant’s spike did not enhance cell fusion in the two Vero cell lines.

In summary, the study suggests that the specific change at the furin cleavage site in the Alpha variant’s spike protein may increase its cleavage. But it does not boost the variant’s ability to infect or replicate in human cells.

As Dr. Whittaker notes, the furin cleavage site may, in fact, “be relatively inconsequential.”

The Delta variant, which was first identified in India in late 2020, was found to be more contagious than previous variants of concern, including the Alpha variant. The Delta variant also caused more severe disease.

Like the Alpha variant, the Delta variant has a mutation at the furin cleavage site. However, this mutation is different from Alpha’s and is associated with increased cleavage by furin.

This mutation of Delta’s is also associated with enhanced cell entry and cell-to-cell fusion, and increased disease severity.

However, the Omicron variant that emerged in November 2021 has an identical mutation to the Alpha variant.

Dr. Whittaker says: “Omicron went back to square one. It returned to the same genetic change in the furin cleavage site that Alpha had. It essentially took a large step back in its evolutionary trajectory as a disease agent.”

“Alpha would cause cells to fuse. Delta would fuse cells even more…. but then Omicron comes along, and its host cells aren’t fusing at all. It has gone completely backward,” he explains.

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